RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof
A technology of vibrio alginolyticus and a kit is applied in the field of detecting vibrio alginolyticus in shellfish aquatic products, and achieves the effects of wide application range, high amplification efficiency and short reaction time
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Embodiment 1
[0053] This embodiment provides the RPA primer pair and the amplification internal standard sequence and its application.
[0054] The screening method for the RPA primer pair is as follows: Aiming at the Vibrio alginolyticus collagenase gene (GenBank ID: CP012739.1), multiple RPA primers were designed and screened in large quantities, and their specificity, sensitivity, and the interaction between primers were integrated. , and the adaptability of the primers used to the RPA amplification kit, and finally screened out the following RPA primer pairs with good specificity, good repeatability and high sensitivity that can detect Vibrio alginolyticus:
[0055] collagenase collagenase-F: 5'-GCTTATCGAGTCGATGCGCCTGACAAGTGATC-3' (SEQ ID NO: 1);
[0056] collagenase collagenase-R: 5'-TCATAAGGGTCCCGTATGCGTACTTGCTAT-3' (SEQ ID NO: 2);
[0057] Design and synthesis of amplified internal standard sequence: In order to avoid homologous interference of similar bacteria and individual nucle...
Embodiment 2
[0062] This embodiment provides a kit and application thereof, the kit comprising:
[0063] (1) SEQ ID NO: 1, SEQ ID NO: 2 (both from Example 1), and a plasmid containing the amplified internal standard sequence shown in SEQ ID NO: 3;
[0064] (2) DNA extraction reagents;
[0065] (3) tube containing freeze-dried enzyme powder;
[0066] (4) rehydration buffer;
[0067] (5) Magnesium acetate solution.
[0068] The above tubes containing lyophilized enzyme powder, rehydration buffer (Rehydration Buffer) and magnesium acetate solution (280mmol / L) are reagents from the RPA amplification kit TwistAmp Basic kits (TwistDX company, product number TABAS03KIT).
[0069] The DNA extraction reagent is the reagent included in the animal genome DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. whose article number is DP432 and the bacterial genome DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. whose article number is DP302. .
[0070] Construction of th...
Embodiment 3
[0076] This embodiment provides a method for detecting or assisting the detection of Vibrio alginolyticus, such as detecting or assisting in detecting whether a biological sample is infected with Vibrio alginolyticus, or detecting or assisting in detecting whether the pathogen is or is a candidate for Vibrio alginolyticus, the method The primer pair of Example 1 or the kit of Example 2 were used.
[0077] Above-mentioned method comprises the steps:
[0078] Step 1: RPA-IAC amplification
[0079] Using the DNA of biological samples or pathogenic bacteria as a template, add the plasmid containing the amplified internal standard sequence shown in SEQ ID NO: 3 in Example 2, and use the primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 to perform RPA -IAC amplification to obtain the RPA-IAC amplification product, while setting a blank control (the template is ultrapure water).
[0080] The preparation method of the RPA-IAC amplification system is as follows: Add 29.5 μL of ...
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